Labelled digoxin derivatives for radioimmunoassay

ABSTRACT

Lysyl tyrosine methyl ester can be coupled to digoxin (digoxigenin tridigitoxoside) by reacting the Epsilon -amino group of the lysine residue with the opened terminal sugar group of digoxin to yield a new digoxin derivative. The derivative can be labelled at the benzyl ring of the tyrosine residue with 125I to space the label away from the antigenic moiety of the digoxin derivative. The spaced label minimizes deleterious effects of gamma radiation on the antigenic moiety, yields a labelled digoxin derivative having an affinity for anti-digoxin antibodies about equal to that of unlabelled digoxin, and facilitates the radioimmunoassay of digoxin using gamma scintillation counters.

United States Patent [191 Piasio et al.

[ Dec. 9, 1975 LABELLED DIGOXIN DERIVATIVES FOR RADIOIMMUNOASSAY [73] Assignee: Corning Glass Works, Corning,

[22] Filed: Mar. 1, 1974 [21] Appl. No.: 447,249

[52] US. Cl. 260/210.5; 424/182; 424/1 [51] Int. Cl. C070 173/02 [58] Field of Search 260/2105, 211 R, 211.5

[56] References Cited UNITED STATES PATENTS Hawker; C. D., Radioimmunoassay, Analytical Chem, Vol. 45, No. 11, p. 882, Sept., 1973.

Primary Examiner.lohnnie R. Brown Assistant Examiner-Cary B. Owens Attorney, Agent, or Firm-James A. Giblin; Clinton S. Janes, Jr.; Clarence R. Patty, Jr.

[57] ABSTRACT Lysyl tyrosine methyl ester can be coupled to digoxin (digoxigenin tridigitoxoside) by reacting the e-amino group of the lysine residue with the opened terminal sugar group of digoxin to yield a new digoxin derivative. The derivative can be labelled at the benzyl ring of the tyrosine residue with i to space the label away from the antigenic moiety of the digoxin derivative. The spaced label minimizes deleterious effects of gamma radiation on the antigenic moiety, yields a labelled digoxin derivative having an affinity for antidigoxin antibodies about equal to that of unlabelled digoxin, and facilitates the radioimmunoassay of digoxin using gamma scintillation counters.

5 Claims, 9 Drawing Figures I llljllllfo I lllllllJ ng/ml Fig. 2

Dec. 9, 1975 Sheet 1 of3 |||||||1| l llllllll 0 L0 ng/ml F/g.

US. Patent 4 m mxm m m w M 3 m US. Patent Dec. 9, 1975 Sheet 3 of3 3,925,355

3 TRACER x g 1 TRACER O I l I Illlll l l lllllll m w I IO ng/ml LABELLED DIGOXIN DERIVATIVES FOR RADIOIMMUNOASSAY BACKGROUND OF THE INVENTION 1. Field This invention relates generally to the field of radio assays and specifically to a radioactively labelled digoxin derivative useful in the radioimmunoassay (RIA) of digoxin.

Radioimmunoassay is a term used to describe any of several methods for determining very small concentrations of substances (especially in biological fluids),

consideration has been given to labelling digoxin with which methods are based on the use of radioactively labelled substances which can form immunochemical complexes with antibodies specific to that substance. The RIA of a substance for which there exists antibodies is based on the observation that a known amount of that substance (which has been radioactively labelled) will tend to compete equally with the unknown amount of the substance (unlabelled) for a limited number of complexing sites on antibodies specific to the substance. Thus, the RIA of a given substance is performed as follows: a known amount of the substance (labelled) and the unknown amount of the substance (unlabelled) are incubated with anti-substance antibodies. During incubation, there are formed immunochemical complexes of both antibody-substance (labelled) and antibody-substance (unlabelled). After an appropriate incubation period, the immunochemical complexes are removed from the reaction solution. Then, radioactivity measurements (counts) are taken of either the removed complexes or the remaining solution. The counts can then be used to determine the unknown concentration by relating the counts to a standard curve prepared beforehand using known amounts of unlabelled substance.

An essential reagent for the RIA of a given substance is the labelled substance. Ideally, the labelled substance (or a labelled derivative of the substance, having complexing ability) has an affinity for the anti-substance antibodies which is about equal to the affinity of the unlabelled substance.

2. Prior Art Digoxin, sometimes described as diogoxigenin tridigitoxoside, is a cardiac glycoside which is used as a heart stimulant. The compound is used in very small amounts and the difference between therapeutic and toxic amounts is very slight. Hence, it is extremely important to have a reliable method for accurately determining digoxin concentrations in serum or plasma samples. To data, RIA offers the only practical method for determining serum digoxin concentrations since the clinically significant concentration range of the substance is very low (e.g. approximately 0.5 to about 5 nanograms per milliliter of biological fluid or serum).

A very common method for labelling digoxin for use in the RIA of digoxin involves tritiating a sample of digoxin. The tritiation replaces H atoms with H atoms on the digoxin molecule and the replacement can be either random or specific depending on the tritiation method used. Since tritium has a relatively long radiation half life (e.g. about 12.3 years), I l-labelled digoxin has the advantage of having a relatively long shelf-life. However, since H is a relatively weak beta radiation emitter, the use of H-labelled digoxin commonly requires that added processing steps and materials be used prior to counting with a liquid scintillation counter. For exan isotope of iodine such as I which emits a more energetic gamma radiation. In using a label such as L however, care must be taken to assure that the label (or tag) is spaced some distance away from the antigenic moiety of the digoxin molecule so that the antigenic moiety (or complexing portion) of the digoxin will not be irreparably modified by radiation from the label. If such modification occurs, there can result a loss in affinity of the labelled digoxin for the anti-digoxin antibodies. Such a loss in affinity affects the accuracy and reliability of a RIA of digoxin since an accurate RIA presupposes an equal competition between labelled and unlabelled digoxin for a limited number of complexing sites on anti-digoxin antibodies.

' Various techniques for labelling certain steroid-albumin conjugates with I for use as tracers in radioimmunoassays are disclosed by S. L. Jeffocate et al. in Clinica Chimica Acta, 43, 343-349 (1973). Methods for conjugating digoxin to the amino groups of lysine residues in human serum albumin are disclosed by T. W. Smith et al., in Biochemistry, 9, No. 2, 331-337 1970) and by v. P. Bulter et al. in Proc. N.A.S., 57, 71-78 (1967). A method of labelling a digoxin derivative with 1 is disclosed by S. Gutcho et al. in Clin. Chem. 19/9, 1058-59 (1973). In that disclosure, 3-O- succinyl digoxigenin 1) tyrosine is used as the labelled substance and that substance is used in comparative tests with H-digoxin. As disclosed in that article, however, the affinity of the labelled derivative for antidigoxin antibodies did not fully correlate with the affinity of unlabelled digoxin.

We have found that a digoxin derivative can be prepared which can be readily labelled with 'I and the labelled derivative has an affinity for anti-digoxin antibodies about equal to that of unlabelled and tritiated digoxin. The derivative and methods for preparing, labelling, and using it are described more fully hereunder.

SUMMARY OF THE INVENTION Our digoxin derivative consists of the compound formed by reacting the e-amino group on the lysine residue of lysyl tyrosine methyl ester with the opened terminal sugar residue of digoxin under reaction conditions described in detail hereunder. Once the lysyl tyrosine methyl ester is coupled through the terminal sugar residue, the benzyl ring of the tyrosine residue can be iodinated with thereby providing a spaced gamma label and a labelled digoxin derivative which has an affinity for digoxin antibodies about equal to digoxin.

BRIEF DESCRIPTION OF THE FIGURES FIGS. 1-8 illustrate standard curves generated using our labelled digoxin derivatives which had been stored under varying conditions for various periods of time.

FIG. 9 compares standard curves generated using our labelled digoxin derivative and tritiated digoxin.

SPECIFIC EMBODIMENTS Preparation of Lysyl Tyrosine Methyl Ester In preparing the product we used L-amino acid residues although it is thought the D-residues could also be successfully used. Our preferred method of preparing the above-described compound is as follows: dissolve 2.3 grams of tyrosine methyl ester in 20 ml. of dimethylformamide (DMF) in a 100 ml. round-bottom flask. Stir on a magnetic stirrer. Adjust the pH to 8.0 with triethylamine (TEA). Add 5.0-1 grams of a-t BOC-e-CBZ L-lysine ONP [oz-tertiary butyloxy carbonyl e-(carbobenzoxy)-L-lysine p-nitrophenylester] I previously dissolved in 10 ml. of DMF. Mix for 24 hours. Maintain the pH at 8.0 with the TEA. When coupling is complete, concentrate off the DMF. The product should form a thick gel. Dissolve the product in ethyl acetate (100 ml.). Add 300 ml. of 10% ammonium hydroxide to the ethyl acetate solution. Stir on a magnetic stirrer for 30 minutes. Using a separatory funnel, separate the two layers. Wash the ethyl acetate layer with distilled water. Dry the ethyl acetate over anhydrous sodium sulfate. Filter away from the sodium sulfate and concentrate the ethyl acetate to an oil. Dissolve the product in cold 100% trifluoroacetic acid (TFA) and stir for I O=C fifteen minutes. Evaporate the TFA to dryness. Dissolve the residue in 15 ml. of glacial acetic acid. Add 15 Jiiig ml. of cold 4N hydrobromic acid and mix for two hours. Add 300 ml. of ethyl ether and mix. A precipitate will appear. Filter away the precipitate from the filtrate. Place the precipitate under vacuum for 24 hours. Dissolve the peptide in acetic acid and lyophilize. The compound has the following structure:

Coupling of Lysyl Tyrosine Methyl Ester to Digoxin:

Digoxin has the following chemical structure:

Our general method for coupling the lysyl tyrosine methyl ester to digoxin involves opening the terminal sugar residue on the digoxin with sodium metaperio date to form:

At a pH of 9.0-9.5, the e-amino group of the lysine residue of the lysyl tyrosine methyl ester is coupled to the terminal open sugar residue on the digoxin to form:

CH CH3 HOC With the addition of sodium borohydride, the above compound is reduced to:

CH c. 3

Our actual steps used in preparing the above compound are as follows:

First, 436 mg (0.56 mole) digoxin is suspended in 20 ml. of absolute ethanol at room temperature in a 250 ml. Erlenmeyer flask. Then, 20 ml. of 0.1M sodium metaperiodate added. After 25 minutes, 0.6 ml. of 1M ethylene glycol is added. Five minutes later, the reaction mixture is added to 900 mg of the lysyl tyrosine methyl ester in 20 ml. of an aqueous buffer solution which had previously been adjusted to pH 9.5 with 5% K CO The pH is maintained at 9.0-9.5 with the K CO for 45 minutes. Then 300 mg of sodium borohydride freshly dissolved in 20 ml. of water, is added. Three hours later, the pH is raised to 8.5 by the addition of 1M ammonium hydroxide. The entire reaction mixture is stirred overnight. After the above period the pH is reduced to 4.5 by the addition of 0.1N HCl. After 4 hours at 4C. the entire reaction mixture is centrifuged and the precipitate dissolved in 10% acetic acid and lyophilized. The product is stored at 20C.

Iodinization of the Digoxin Derivative:

The iodinization is carried out according to the general procedure suggested by Hunter and Greenwood in Nature, Vol. 194, 495-6 (1962).

One millicurie of Na I is reacted with 10-20 p. gms of the digoxin derivative in a small vial or tube. Chloramine T is added and quickly mixed. After 8-10 seconds sodium metabisulfite (100 ,u. gms) is added and mixed also quickly. 500 p. gms of potassium iodide is added and mixed. The entire reaction mixture is transferred to a DEAE Sephadex column and eluted with 0.1M Na phosphate buffer. Three ml. aliquots are collected. Each aliquot is assayed for binding with digoxin antibody. Tubes with highest binding activity are pooled and aliquoted out and stored at appropriate temperature. In all cases the reagents are dissolved in 0.1M sodium phosphate pH 7.4.

In following the above procedure the digoxin derivative is labelled at C or C and C of the benzyl ring of the tyrosine residue, depending generally on duration of the iodination procedure. The actual percentages of monoand di-iodinated derivative can be readily determined by conventional strip scanning techniques. Thus,

signed to the present assignee. The assay conditions were as follows: a -minute incubation period was followed by a 5-minute centrifugation at 2,500 rpm. After centrifugation, the supernatent was removed by aspiration (with no decantation).

FIG. 1 illustrates digoxin standard curve using 1 digoxin that has been stored at 4C. for 26 days.

FIG. 2 illustrates digoxin curve using I digoxin l0 stored at room temperature for 26 days.

20 has been stored at room temperature for 14 days.

FIG. 7 illustrates digoxin curve using digoxin curve using I digoxin that has been stored at 37C. for 14 days.

FIG. 8 illustrates digoxin curve using I digoxin that the final iodinated derivative can have either or both of 25 has been stored in lyophilized form for 14 days. The

the following structures:

(mono-) Preparation of Standard Curves:

Samples of the labelled digoxin derivative were used to prepare a series of standard curves which reflected the complexing ability of the derivatives after storage at different temperatures and for varying periods of time. These curves are indicated in FIGS. 1-8. All curves were prepared with anti-digoxin antiserum obtained from Biospheres, Inc. of Miami, Fla. The anti-serum had been immobilized by chemical coupling through a silane coupling agent to porous glass particles. The immobilization procedure is described in copending patent application Ser. No. 447,252, filed of even date, entitled, Solid Phase Radioimmunoassay, and asbuffers used in iodinating thederivatives used in preparing the standard curves of FIGS. 4-8 was PBS/BSA NaN Comparison with other I-Digoxin Derivatives and Tritiated Digoxin Using essentially the same assay conditions, our 1- digoxin derivative was used to prepare a standard curve essentially identical to that generated with tritiated digoxin obtained from New England Nuclear Corp. The curves are compared in FIG. 9 where I Tracer designates the curve obtained with our I-digoxin derivative and H Tracer represents the curve generated with tritiated digoxin.

' -CO I OH ef c v2. zs O I CH CH CH; H Hz? a o o H HCH H $-NHC-$HCH CH -CH;-CH; -n-c' l 0 NH: H OH OH I as In FIG. 9 it can be seen that our labelled digoxin de- 4. A method of making a digoxin derivative comprisrivative that has an affinity for anti-digoxin antibodies ing the steps of: closely approximating that of tritiated digoxin a. reacting a solution of digoxin with sodium metaperiodate to form a first digoxin derivative; b. reacting the derivative formed in step (a) with lysyl tyrosine methyl ester to form a second digoxin de- We claim: rivative;

c. reacting the second digoxin derivative with a solul. A digoxin derivative having the following chemical tion of sodium borohydride to reduce the second structure: digoxin derivative.

CH OH d6 3 CH, CH CH 0H H c o l O O H I ll H CH H H C-NH-C-CH-CH CH CH CH ---c 0 O I l H OH OH 0 NH:

O I N 2. A digoxin derivative having the following chemical 5. The method of claim 4 which includes the addistructure: tional and subsequent step of iodinating the tyrosine 30 (:H OH

I125 CH: CHBO CH 0 H 0 Hz? H cH CH CH cH fiwmm H (E-NH-C-(i z z z 2 H OH OH o= NH:

3. A digoxin derivative having the following chemical residue with structure: 5 

1. A DIGOXIN DERIVATIVE HAVING THE FOLLOWING CHEMICAL STRUCTURE:
 2. A digoxin derivative having the following chemical structure:
 3. A digoxin derivative having the following chemical structure:
 4. A method of making a digoxin derivative comprising the steps of: a. reacting a solution of digoxin with sodium metaperiodate to form a first digoxin derivative; b. reacting the derivative formed in step (a) with lysyl tyrosine methyl ester to form a second digoxin derivative; c. reacting the second digoxin derivative with a solution of sodium borohydride to reduce the second digoxin derivative.
 5. The method of claim 4 which includes the additional and subsequent step of iodinating the tyrosine residue with 125I. 